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Fig. 10. ER and Pit-1 mobility over PRL array. (A) Representative time-lapse images of photobleaching and recovery of GFP-ER-targeted arrays (FRAP). FRAP was used to examine GFP-ER at the PRL array and in the surrounding nucleoplasm (images not shown). PRL-HeLa cells transiently expressing GFP-ER were treated with vehicle or ligands at 10 nM for 2 hours. Pre-bleach and bleach refer to cells before and after photobleaching. Time in seconds after bleaching is indicated. (B) Representative time-lapse images of nuclear photobleaching and loss of GFP-ER from targeted arrays (inverseFRAP). In these experiments, PRL-HeLa cells transiently expressing GFP-ER were treated with E2 (Estrogen) or 4HT (Tamoxifen). Pre and Post refer to images acquired prior to and immediately post photobleaching; time after post-bleach is indicated in seconds. (C) PRL-HeLa cells transiently expressing GFP-Pit-1 were analyzed by FRAP as described in A. (D) FRAP recovery curves are shown for nucleoplasm (left) and PRL array (right) in cells transiently expressing GFP-ER and treated with vehicle (No ligand), E2, 4HT (Tam) and ICI. The initial fluorescence immediately before the bleach was normalized to 1 and the curve starts from the time point immediately after the bleach. Note that non-ligand-dependent interactions greatly slowed the recovery of receptor over the PRL array compared with nucleoplasm, but E2 did not additionally decrease recovery times of GFP-ER at the array. 4HT lead to faster recovery both on and off the array. ICI, as expected, immobilized GFP-ER in the nucleus. (E) iFRAP loss curves were obtained in a similar manner, except that prebleach values were retained. PRL-HeLa cells transiently expressing GFP-ER were treated with vehicle, E2 or 4HT (10 nM) prior to photobleaching. The fluorescence at the PRL array was averaged over a 60-second time frame. The initial fluorescence immediately after photobleaching was normalized to 1 and relative fluorescence before bleaching was set to zero. k_off was slower with E2 than no ligand or 4HT. n=30 for FRAP analysis under each condition.

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